1. Field of the Invention
The present invention relates to a method for constructing a DNA library having increased proportion of a desired nucleic acid by removing, from an original DNA library, a nucleic acid other than the desired nucleic acid using RecA protein.
2. Description of the Related Art
DNA libraries, particularly cDNA libraries, are very useful tools for gene cloning. To date, various genes have been cloned from cDNA libraries. A cloned gene is used to determine an amino acid sequence of a protein encoded by the gene and to produce a large amount of the protein in bacterial or yeast cells, as well as to determine its nucleotide sequence.
However, cDNAs that can be easily cloned from a cDNA library are restricted to those whose template mRNAs are abundantly expressed in a cell. It is getting harder to clone a novel cDNA with high efficiency, because cDNAs easy for cloning have now mostly been cloned.
In order to efficiently clone novel cDNAs from a cDNA library, it is necessary to remove cDNAs already cloned from the library. For this purpose, the following prior arts have been devised.
Subtractive hybridization has been used primarily for this purpose. In this method, mRNAs are harvested both from cells (or tissues) expressing genes of interest and those not expressing them. Then cDNAs are synthesized from the mRNAs in either cell. Through hybridization of the synthesized cDNAs with the mRNAs, cDNAs that are present in both cells are selectively removed. This enables enrichment and isolation of genes specifically expressed in a particular tissue or a cell.
“Genome Res. 1996 Sep: 6(9): pp. 791-806” discloses subtractive hybridization using a hydroxyapatite column. In this method, primers from vector-derived sequences are elongated using a single-stranded DNA library as templates. After denaturation and annealing, DNAs that form a double strand again are specifically removed by the column. Since probability of annealing is dependent on concentration, abundant clones are preferentially removed.
However, this method can only be applied to relatively short cDNAs approximately 0.4-2.5 kb in size, because non-specific hybridization can occur when cDNA libraries are used that include long sequences with an insert size greater than 3 kb. Long sequences are often found to be a functionally important gene encoding a multifunctional protein or a protein with complex conformation. Therefore, it is a major drawback of this method that it cannot be applied to a library containing a long sequence. Furthermore, this method cannot distinguish even short cDNAs, if their sequences are derived from an identical gene and have common sequences at 3′ and 5′ ends only, not in their central regions.
Other method widely used for a similar purpose is differential hybridization.
In this method, cDNA probes are synthesized with mRNAs prepared both from control cells and cells of interest from which a specific gene is obtained. A cDNA library generated from the cells of interest is then plated, and colonies on the same plate are replica-plated onto two filters. For one filter, hybridization is performed with cDNA probes from the cells of interest. For the other, hybridization is performed with cDNA probes from the control cells. cDNAs specific for the cells of interest can be detected by comparing the results.
However, in this method, differences in hybridization between two filters must be compared from colony to colony. Accordingly, it is difficult to handle numerous colonies by the method. This method is thus not suitable for reconstitution of a whole library. This method has an additional drawback of being time-consuming for checking many possible pseudo-positive or pseudo-negative signals.
To overcome such drawbacks of this method, “Methods in Enzymology 1995: 254: pp. 304-321” discloses differential display method, which is a combination of conventional differential hybridization and polymerase chain reaction (hereinafter referred to as “PCR”). However, this method can detect a difference in a pattern only when the difference in an expression level is significant. In addition, it is necessary to select clones by any method based on a PCR product, because this method cannot produce clones directly.